With the installation of the Water Recovery System (WRS) during mission STS-126 in 2008, the International Space Station (ISS) added the capability to recover clean water for reuse from crewmember urine and atmospheric humidity condensate, including EVA (Extravehicular Activity) wastes. The ability to collect, store and process these waste streams is required to increase potable water recovery and support the ISS crew augmentation planned for 2009. During ground testing of the Urine Processing Assembly (UPA), one of two primary component subsystems that comprise the WRS, significant fouling was repeatedly observed in stored urine pretreated with 0.56% of chromium trioxide and sulfuric acid. During initial observation, presumptive microbiological growth clogged and damaged flight-rated hardware under test as part of a risk-mitigation Flight Experiment (FE). The objective of this report is to characterize the extent of biological contamination in the pre-treated urine (pH<2) and to isolate and identify any fungi or bacteria capable of growth at pH<2.5 in the presence of chromium trioxide. This data will be used to quantify the potential risk of microbial growth in pre-treated wastewater on orbit, and to identify the microbial control strategies necessary to prevent microbiologically induced damage to Environmental Control and Life Support (ECLS) system hardware used for water recovery. Cultivation and molecular based methods were used to recover viable microorganisms and DNA from pretreated urine samples and to determine total cell density by direct microscopic count. Total cell density in the urine quantified by microscopic observation revealed >105 cells per milliliter. Multiple selective media were used to recover bacteria and fungi from pretreated urine samples. DNA was extracted and purified from viable microorganisms recovered on solid media, amplified and sequenced to provide sequence-based species identification of recovered bacteria and fungi. In addition, total DNA recovered directly from pretreated urine samples was assayed by Terminal Restriction Fragment Length Polymorphism (TRFLP) to profile fungal populations that could not be cultivated on selected agar media. TRFLP fungal profiling identified the presence of DNA from five different fungi in the fungal mat community recovered from the UPA in ground test. A single fungal species was recovered from the fungal mat on selective media and typed by sequence-based identification as Cladosporium cladosporiodes. A single bacterial species was recovered on selective media from the pretreated urine and identified as Paenibacillus curdlanolyticus. Both microorganisms are routinely isolated as environmental air contaminants, but their growth at low pH in the presence of chromium trioxide has not been confirmed. Based on relative biomass, fungal growth was the primary factor contributing to biofouling and damaged hardware in the UPA.